The pUL37 tegument protein guides alphaherpesvirus retrograde axonal transport to promote neuroinvasion

A hallmark property of the neurotropic alpha-herpesvirinae is the dissemination of infection to sensory and autonomic ganglia of the peripheral nervous system following an initial exposure at mucosal surfaces. The peripheral ganglia serve as the latent virus reservoir and the source of recurrent infections such as cold sores (herpes simplex virus type I) and shingles (varicella zoster virus). However, the means by which these viruses routinely invade the nervous system is not fully understood. We report that an internal virion component, the pUL37 tegument protein, has a surface region that is an essential neuroinvasion effector. Mutation of this region rendered herpes simplex virus type 1 (HSV-1) and pseudorabies virus (PRV) incapable of spreading by retrograde axonal transport to peripheral ganglia both in culture and animals. By monitoring the axonal transport of individual viral particles by time-lapse fluorescence microscopy, the mutant viruses were determined to lack the characteristic sustained intracellular capsid motion along microtubules that normally traffics capsids to the neural soma. Consistent with the axonal transport deficit, the mutant viruses did not reach sites of latency in peripheral ganglia, and were avirulent. Despite this, viral propagation in peripheral tissues and in cultured epithelial cell lines remained robust. Selective elimination of retrograde delivery to the nervous system has long been sought after as a means to develop vaccines against these ubiquitous, and sometimes devastating viruses. In support of this potential, we find that HSV-1 and PRV mutated in the effector region of pUL37 evoked effective vaccination against subsequent nervous system challenges and encephalitic disease. These findings demonstrate that retrograde axonal transport of the herpesviruses occurs by a virus-directed mechanism that operates by coordinating opposing microtubule motors to favor sustained retrograde delivery of the virus to the peripheral ganglia. The ability to selectively eliminate the retrograde axonal transport mechanism from these viruses will be useful in trans-synaptic mapping studies of the mammalian nervous system, and affords a new vaccination paradigm for human and veterinary neurotropic herpesviruses

Are local climate adaptation policies credible? A conceptual and operational assessment framework

After the Paris Agreement that put stronger emphasis on the development of climate change adaptation policies and on the definition of financing mechanisms, there is a patent need to track whether actual planning efforts are proving sufficient. This entails the development of assessment methods and metrics as plans are drafted and actions implemented. To this end, this paper explores the concept of credibility as a critical issue in climate policy and develops an Adaptation Policy Credibility (APC) conceptual and operational assessment framework for helping to allocate public funding and private investments, and for implementing and catalysing climate policy. Through a pilot testing in four early-adopting cities (Copenhagen, Durban, Quito and Vancouver), a clear potential for large-n tracking and assessment exercises of local climate adaptation plans is envisaged. The APC approach might also be useful to guide individual cities that aim to improve their adaptation planning and policy-making processes

The pUL37 tegument protein guides alphaherpesvirus retrograde axonal transport to promote neuroinvasion

A hallmark property of the neurotropic alpha-herpesvirinae is the dissemination of infection to sensory and autonomic ganglia of the peripheral nervous system following an initial exposure at mucosal surfaces. The peripheral ganglia serve as the latent virus reservoir and the source of recurrent infections such as cold sores (herpes simplex virus type I) and shingles (varicella zoster virus). However, the means by which these viruses routinely invade the nervous system is not fully understood. We report that an internal virion component, the pUL37 tegument protein, has a surface region that is an essential neuroinvasion effector. Mutation of this region rendered herpes simplex virus type 1 (HSV-1) and pseudorabies virus (PRV) incapable of spreading by retrograde axonal transport to peripheral ganglia both in culture and animals. By monitoring the axonal transport of individual viral particles by time-lapse fluorescence microscopy, the mutant viruses were determined to lack the characteristic sustained intracellular capsid motion along microtubules that normally traffics capsids to the neural soma. Consistent with the axonal transport deficit, the mutant viruses did not reach sites of latency in peripheral ganglia, and were avirulent. Despite this, viral propagation in peripheral tissues and in cultured epithelial cell lines remained robust. Selective elimination of retrograde delivery to the nervous system has long been sought after as a means to develop vaccines against these ubiquitous, and sometimes devastating viruses. In support of this potential, we find that HSV-1 and PRV mutated in the effector region of pUL37 evoked effective vaccination against subsequent nervous system challenges and encephalitic disease. These findings demonstrate that retrograde axonal transport of the herpesviruses occurs by a virus-directed mechanism that operates by coordinating opposing microtubule motors to favor sustained retrograde delivery of the virus to the peripheral ganglia. The ability to selectively eliminate the retrograde axonal transport mechanism from these viruses will be useful in trans-synaptic mapping studies of the mammalian nervous system, and affords a new vaccination paradigm for human and veterinary neurotropic herpesviruses

The pUL37 tegument protein guides alpha-herpesvirus retrograde axonal transport to promote neuroinvasion.

A hallmark property of the neurotropic alpha-herpesvirinae is the dissemination of infection to sensory and autonomic ganglia of the peripheral nervous system following an initial exposure at mucosal surfaces. The peripheral ganglia serve as the latent virus reservoir and the source of recurrent infections such as cold sores (herpes simplex virus type I) and shingles (varicella zoster virus). However, the means by which these viruses routinely invade the nervous system is not fully understood. We report that an internal virion component, the pUL37 tegument protein, has a surface region that is an essential neuroinvasion effector. Mutation of this region rendered herpes simplex virus type 1 (HSV-1) and pseudorabies virus (PRV) incapable of spreading by retrograde axonal transport to peripheral ganglia both in culture and animals. By monitoring the axonal transport of individual viral particles by time-lapse fluorescence microscopy, the mutant viruses were determined to lack the characteristic sustained intracellular capsid motion along microtubules that normally traffics capsids to the neural soma. Consistent with the axonal transport deficit, the mutant viruses did not reach sites of latency in peripheral ganglia, and were avirulent. Despite this, viral propagation in peripheral tissues and in cultured epithelial cell lines remained robust. Selective elimination of retrograde delivery to the nervous system has long been sought after as a means to develop vaccines against these ubiquitous, and sometimes devastating viruses. In support of this potential, we find that HSV-1 and PRV mutated in the effector region of pUL37 evoked effective vaccination against subsequent nervous system challenges and encephalitic disease. These findings demonstrate that retrograde axonal transport of the herpesviruses occurs by a virus-directed mechanism that operates by coordinating opposing microtubule motors to favor sustained retrograde delivery of the virus to the peripheral ganglia. The ability to selectively eliminate the retrograde axonal transport mechanism from these viruses will be useful in trans-synaptic mapping studies of the mammalian nervous system, and affords a new vaccination paradigm for human and veterinary neurotropic herpesviruses

pUL37 R2 is essential for HSV-1 neuroinvasion.

<p><b>(A)</b> Diagram of the neuroinvasive route examined in mice following inoculation of a scarified cornea with HSV-1. <b>(B)</b> Representative images of the cornea (2 dpi), trigeminal ganglia (6 dpi), and principal sensory trigeminal nuclei (Pr5; 6 dpi), following corneal inoculation with wild-type or R2-mutant HSV-1. Infected cells were visualized by virtue of a pUL25/mCherry fluorescent capsid reporter encoded by the viruses (scale bars for trigeminal ganglia and Pr5 are 500 μm; cornea scale bar is 1000 μm). <b>(C)</b> Mice were infected on both eyes with untagged wild-type (WT) or R2-mutant (R2) HSV-1 following corneal scarification. Viral titers in the tear films were independently determined from each eye by swabbing at the indicated day post-infection (dpi). At 4 days post corneal inoculation, the combined titer of the left and right trigeminal ganglia and of the whole brain were determined. The mean titer of each data set is indicated by a red bar (5 mice per virus; *, p < 0.05 based on a two-tailed unpaired <i>t</i> test). <b>(D)</b> qPCR was preformed using primers directed against the HSV-1 UL35 gene. Trigeminal ganglia and brain samples were collected at 4 days post infection and scored as positive for viral DNA if the threshold cycle (Ct) value was below the average Ct value of the water controls by more than two standard deviations (inset). Amplification curves for both HSV-1 WT and R2-mutant infected samples are shown.</p

Mutation of PRV R2 results in a post-entry microtubule-dependent delay in gene expression.

<p><b>(A)</b> Percent of PRV internalized into PK15 epithelial cells prior to extracellular virion inactivation with citrate at indicated times post-infection. Five experimental replicates are shown for each virus, with each representing average values from duplicate infections. <b>(B)</b> Expression of the PRV immediate early gene IE180 was quantified by qRT-PCR and normalized to expression of the host S28 rRNA at 4 hpi with the wild-type (WT) and R2 mutant (R2). Where indicated, 9 μM of nocodazole was added 1 hr prior to infection and maintained throughout the infection. All values were plotted relative to levels observed during the untreated wild-type (WT) infection. Three independent experiments were performed with each experimental replicate performed in triplicate. Values are expressed as mean ± s.e.m. (****, p < 0.0001; ns, not significant based on two-tailed unpaired <i>t</i> test).</p

pUL37 R2 is essential for sustained retrograde motion during ingress.

<p>Dorsal root ganglion (DRG) sensory neuron explants were infected in 2 ml of media with 3.5 x 10⁷ PFU/ml of PRV (WT and R2 mutant) or 1.3 x 10⁷ PFU/ml of HSV-1 (WT and R2 mutant). Viruses encoded a pUL25/mCherry or pUL25/GFP fusion to provide imaging of individual capsids in living cells. Capsid axonal transport was recorded by time-lapse fluorescence imaging between 3–4 hpi. More than 90 capsids were analyzed per experiment across three biological replicates. <b>(A)</b> Fraction of time that individual wild-type (WT) and R2-mutant (R2) capsids moved retrogradely (white), anterogradely (gray), or were motionless (black). Error bars are s.d. <b>(B)</b> Net displacement of capsids over a period of 10 seconds. Positive values indicate movement towards neuronal soma (retrograde displacement). Error bars are s.d. (****, p < 0.0001 based on two-tailed unpaired <i>t</i> test). <b>(C)</b> Representative kymographs of axonal transport in neurons. Distance and time are represented on the x and y axis respectively. <b>(D)</b> Delivery of capsids to nuclear rims at 3–4 hpi following co-infection of DRGs with a pUL25/GFP tagged wild-type (WT) virus and either WT or R2 mutant virus encoding a pUL25/mCherry capsid tag.</p

Mice immunized with the HSV-1 R2 mutant are protected from HSV-1 neuroinvasion and periocular skin disease.

<p><b>(A)</b> Mice were vaccinated by inoculating the left cornea with 7 μl of the HSV-1 R2 mutant stock (R2; 9 x 10⁷ PFU/ml). Sham vaccinated animals received an equivalent volume of conditioned media. At 14 days post vaccination mice were challenged with 7 μl of wild-type HSV-1 strain F (1.0 x 10⁸ PFU/ml) in the right eye. Mice were euthanized at 4 days post infection and the viral load in the whole ipsilateral trigeminal ganglia and brain were determined. The mean titer of each data set is indicated by a red bar (n.d., not detected). <b>(B)</b> Mice were vaccinated as described in (A), at 14 days post vaccination mice were challenged with 5 μl of wild-type HSV-1 McKrae (6.0 x 10⁸ PFU/ml) in the right eye and monitored for survival. <b>(C)</b> Mice were vaccinated as described in (A), and at 14 days post vaccination mice were challenged in the right eye with wild-type HSV-1 (either strain F or McKrae as indicated). The right eye of vaccinated animals was scored for periocular skin disease at the indicated day post challenge. Scoring was based on previous published criteria [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006741#ppat.1006741.ref056" target="_blank">56</a>]: 0, no lesions; 1, minimal eyelid swelling; 2, moderate eye lid swelling; 3, severe eye lid swelling with no periocular hair loss; 4, eyes swollen shut with minimal ocular discharge and periocular hair loss; 5 eyes swollen shut with severe periocular hair loss and skin lesions. Values are mean disease scores ± s.d. * Mice were euthanized at 5 days post challenge due to pronounced neurological symptoms.</p

The PRV pUL37 R2 region is essential for virulence and can be mutated without causing misfolding of the surrounding protein structure.

<p><b>(A)</b> Kaplan–Meier presentation of mouse survival following intranasal instillation of wild-type PRV (WT) or PRV carrying mutations in the R1, R2, or R3 regions of the pUL37 tegument protein (n = 5 animals for each virus). All viruses encode a mCherry tag fused to the pUL25 capsid protein as previously described [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006741#ppat.1006741.ref030" target="_blank">30</a>,<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006741#ppat.1006741.ref047" target="_blank">47</a>]. <b>(B)</b> The crystal structure of the N-terminal half of the PRV pUL37 R2 mutant (R2; lilac), determined in this work, was overlaid onto the previously determined wild-type structure (WT; beige) [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006741#ppat.1006741.ref047" target="_blank">47</a>] with rmsd 0.5538 Å over 479 aligned residues as determined in Coot [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006741#ppat.1006741.ref093" target="_blank">93</a>]. A close-up view of R2 is shown to the right with the side chains of the five targeted amino acids indicated for wild type and the mutant.</p

Mutation of the HSV-1 pUL37 R2 cluster phenocopies the PRV pUL37 R2 mutant.

<p><b>(A)</b> Amino acid mutations resulting from five codon changes introduced into the UL37 gene of HSV-1 are indicated at top, along with the corresponding mutations of the previously described PRV R2 mutant and the respective positions of the mutations within a sequence alignment of nine alpha-herpesviruses (mutated positions highlighted in red). <b>(B)</b> Wild-type (WT) and R2-mutant (R2) HSV-1 single-step propagation kinetics were determined by counting plaque-forming units harvested from media and Vero epithelial cells (cells) at the times indicated. Viruses used in this study were not modified to encode a fluorescent tag. <b>(C)</b> Fluorescent plaque diameters of WT and R2-mutant HSV-1 encoding mCherry fused to the pUL25 capsid protein were compiled from three independent experiments and plotted as a percentage of the average WT plaque diameter. Error bars are s.d. (****, p < 0.0001 based on a two-tailed unpaired <i>t</i> test).</p